Small and Dangerous: MicroRNA-21 and Blindness

Abstract

Background: Retinal and choroidal neovascularization (RNV and CNV, respectively) are characterized by the inappropriate growth of retinal capillaries that may progress to retinal scarring, detachment and vision loss. MicroRNAs (miRs) are short noncoding RNAs which have been demonstrated to modulate diverse cellular processes such as cell differentiation, proliferation, and apoptosis. Our group and others have shown that miR-21 plays a crucial role in regulating angiogenesis and neovascularization in retina. We have previously shown that activation of STAT3/miR-21 pathway leads to loss of TIMP3 and activation of MMP2 and MMP9. Increased activity of MMP2 and MMP9 in the ischemic retina has been linked to the proteolytic degradation of pigmented epithelial derived factor (PEDF), a key retinal angiostatic factor. Importantly, miR-21 targets peroxisome proliferator-activated receptor alpha (PPARα). PPARα-responsive elements are found in PEDF promoter suggesting that this could be a potential transcription factor for PEDF. The role of miR-21 in regulating PEDF and PPARα in Human Retinal Pigmented Epithelial cells (HuRPE) has never been investigated and is the main goal of the present study. Methods: HuRPE were treated with VEGF at different time points. Transfection of HuRPE cells was performed using a specific miR-21 inhibitor, a miR-21 mimic, and scrambled miRNA as a negative control. Western blot and real-time PCR were used to evaluate the expression of PEDF and PPARα. Luciferase assay was performed to study the interactions between PPARα and PEDF. Results: VEGF treatment of HuRPE cells promoted the expression of miR-21 while PEDF and PPARα expression was down regulated. Further, overexpression of miR-21 decreased PEDF and PPARα expression. Next, we observed that inhibiting miR-21 expression could rescue VEGF-induced down regulation of PEDF and PPARα. To study the specific relationship between PPARα and PEDF, we treated HuRPE cells with siPPARα (inhibition) or PPARα agonist (fenofibrate) (induction). While, inhibition of PPARα expression decreased PEDF expression, PPARα agonist enhanced PEDF 5 expression. Lastly, using a PEDF promoter plasmid we observed that, PPARα could regulate PEDF expression by modulating its promoter activity. Conclusion: Collectively, our data shows that VEGF-mediates induction of miR-21 expression regulates PPARα-PEDF axis and could have a significant role in choroidal neovascularization. This suggests that miR-21 potentially plays a critical role in age-related macular degeneration. Keywords: Retinal Neovascularization, MicroRNA-21, PEDF, and PPARα